TABLE 2.
PCR primers and restriction endonucleases used in the SNP assay for this study
| SNP name | Primer name | Primer sequence (5′ to 3′) | Annealing temp (°C) | Product size (bp) | Enzyme | Restriction resultsa |
|---|---|---|---|---|---|---|
| snp3842359b | MAP_F1 | CACCTGGCCAAGTACTACCA | 63 | 528 | BsmBI | (A) Type C; 528 bp |
| MAP_R1 | GCGATGTCATGATGCTGCTG | (G/C) Type S; 312 and 216 bp | ||||
| snp343677 | TypeS_F | AACACCAGGATCGCGTTCTT | 65 | 511 | AvaII | (G) Type I; 297, 152, and 62 bp |
| TypeS_R | CAATTAGCGGTCGAGTCGTC | (A) Type III; 293 and 218 bp | ||||
| snp50173 | BisonF | GGACGATTACTCGGTTCCAG | 63 | 469 | ApoI | (T) Bison group; 226, 192, and 51 bp |
| BisonR | ACCCGTGTTCGGCTACCT | (G) Cattle group; 277 and 192 bp | ||||
| snp4111202 | SNP4_F | GTCAGAAACATCCCGCCTTC | 65 | 461 | FatI | (G) Type C subgroup A; 284 and 177 bp |
| SNP4_R | GTATTGAGTGAGGCAAGCGG | (C) Type C subgroup B; 461 bp | ||||
| snp3879247 | SNP5_F | GTTGATCGACAGCGAGTGC | 64 | 465 | BlpI/DdelI | (C) 227 and 238 bp |
| SNP5_R | GTGGTGTCCGAGGTGAACTT | (T) 465 bp | ||||
| snp2939977 | SNP6_F | TATCTCCAAGGACGCATTCC | 64 | 516 | NAc | NA |
| SNP6_R | CTGCCATGTCCGTCCTTAAT | |||||
| snp1932058 | SNP7_F | GGCTTGAAACTCCAACTGCT | 63 | 452 | NA | NA |
| SNP7_R | CGTCGTACATCCTCGTGGT | |||||
| snp1327872 | SNP8_F | GCGCTTGTTGTACAGGTTGA | 64 | 528 | AvaI/BsoBI | (G) 292, 171, and 65 bp |
| SNP8_R | TACGACGAAGACCCCGACTA | (T) 463 and 65 bp | ||||
| snp3844632 | SNP9_F | GATCGATGCGGAGCTCGT | 64 | 457 | FatI | (G) 457 bp |
| SNP9_R | TGACAGGAAGGTCCATAGCC | (C) 241 and 217 bp | ||||
| snp1966028 | SNP10_F | GTCGAGGGCTTCCAGGTT | 67 | 427 | SapI/EarlI | (A) 427 bp |
| SNP10_R | GTCTGAGGCCAGCGACAC | (C) 246 and 182 bp | ||||
| snp305277d | SNP11_F | CCATCCCGAGTTCAACAAGT | 64 | 461 | BspMI/BfuAI | (G) 310 and 151 bp |
| SNP11_R | ACTTGTCGGGGTTGTAGCTG | (A) 461 bp | ||||
| snp4339946 | SNP12_F | AACCGCTCAAGGCGAAAG | 64 | 464 | BstEII | (T) 292 and 172 bp |
| SNP12_R | TCCCTTATCTGCGAAGTGCT | (A) 464 bp | ||||
| snp2087274 | SNP13_F | CAGACCGAGCACCTCCTG | 65 | 453 | HpyAV | (C) 453 bp |
| SNP13_R | CCGCGTTGAAGGATCTCAAG | (A) 227 and 226 bp | ||||
| snp1686154 | SNP14_F | GAATCCCCGGAACTGGTG | 65 | 525 | MscI | (G) 525 bp |
| SNP14_R | GCAGTCCAGATAACGGAACG | (A) 284 and 241 bp |
a
Letter in parentheses represents the expected base at the correspondent SNP position.
b
SNP can also distinguish between types I and III; type I has a G, and type III has a C, at base position 3842359, detectable by sequencing the PCR product.
c
NA, not applicable.
d
SNP can also distinguish between two phylogenetic subgroups of bison group isolates (Fig. 3).